Our prior report noted that we had detected the presence of chymotrypsin mRNA in the rat brain. The present work was undertaken to study a variety of peripheral organs (intestinal tract, heart, lung, kidney, liver, spleen, thymus, adrenal, bladder, testis, ovary, and pancreas) using molecular biological and immunocytochemical methods in order to detect chymotrypsin mRNA and immunoreactivity. Using a rat chymotrypsin probe an a ribonuclease protection assay, the presence of chymotrypsin mRNA was detected in the pancreas, stomach, duodenum, jejunum, ovary, and spleen. Northern blot analysis of mRNA poly (A)+ revealed a chymotrypsin message in the stomach, jejunum, spleen, testis and pancreas. Only trace amounts were noted in the thymus and liver. Immunofluorescence microscopy with polyclonal and monoclonal antibodies to human chymotrypsin detected (a) chymotrypsin-like immunoreactivity in endocrine-like cells of the basal third of the villi of the stomach; (b) positive immunoreactivity in some epithelial cells of the duodenum and jejunum. Rare cells were seen in the ileum; (c) positive immunoreactivity in the acini of the exocrine glands of the pancreas. The presence of chymotrypsin in the stomach and intestine suggests that, in addition to pancreatic-derived enzyme, additional chymotrypsin may be elaborated by the gut for protein metabolism. Since we were not able to localize chymotrypsin-like immunoreactivity in the spleen, ovary, and testis, we cannot speculate about the significance of mRNA in these organs.